Proteomic Analysis of Laser-Captured Paraffin ... · A Molecular Portrait of Head and Neck Cancer Progression ... Supplementary data for this article are available at Clinical Cancer

Proteomic Analysis of Laser-Captured Paraffin-EmbeddedTissues:AMolecular Portrait of Head and Neck Cancer ProgressionVyomesh Patel,1Brian L. Hood,2 Alfredo A.Molinolo,1Norman H. Lee,4 Thomas P. Conrads,2

John C. Braisted,5 David B. Krizman,3 Timothy D. Veenstra,2 andJ. Silvio Gutkind1

Abstract Purpose: Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalentcancer amongmenworldwide, is associated with poor prognosis, whichhas improved onlymar-ginally over the past three decades. A proteomic analysis of HNSCC lesions may help identifynovel molecular targets for the early detection, prevention, and treatment of HNSCC.Experimental Design: Laser capture microdissection was combined with recently developedtechniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and anovel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections ofnormal oral epithelium and well, moderately, and poorly differentiated HNSCC were processedfor mass spectrometry and bioinformatic analysis.Results: A large number of proteins expressed in normal oral epithelium and HNSCC, includingcytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cellmaintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, ma-trix degradation, and proteins with tumor suppressive and oncogenic potential, were readilydetected. Of interest, the relative expression of many of these molecules followed a distinct pat-tern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumortissues. Representative proteins were further validated using immunohistochemical studies inHNSCC tissue sections and tissue microarrays.Conclusions:The ability to combine laser capture microdissection and in-depth proteomic anal-ysis of FFPE tissues provided a wealth of information regarding the nature of the proteinsexpressed in normal squamous epithelium and during HNSCC progression, which may allow thedevelopment of novel biomarkers of diagnostic and prognostic value and the identification ofnovel targets for therapeutic intervention in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the sixthmost frequent cancer in the United States and the fourth mostprevalent cancer among men worldwide (1). The prognosis ofHNSCC patients is relatively poor, largely due to the advancednature of the disease at the time of diagnosis. The identificationof the molecular mechanisms underlying HNSCC initiationand progression could aid in the development of newdiagnostic and treatment options for this disease (2, 3). In thisregard, analysis of mRNA transcripts using high-throughputgene array analysis has helped identify numerous moleculesthat may contribute to cancer development (reviewed in ref. 4).A shortcoming of this approach, however, stems from thediscordance between transcript levels and protein abundance ina highly complex and readily changing disease microenviron-ment such as cancer (5). A proteomic analysis is urgentlyneeded, as it allows the comprehensive assessment of thedistinct molecular profile of each cancer type, thus affordingthe opportunity of identifying novel prognostic markers andtherapeutic targets (6).Improved protein extraction protocols (7) combined with

recently developedmass spectrometry (MS) techniques and fullyannotated genomic databases has allowed the identificationof trace amounts of proteins present in complex samples (8).

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Authors’Affiliations: 1Oral and Pharyngeal Cancer Branch, National Institute ofCraniofacial and Dental Research, NIH, Bethesda, Maryland; 2Laboratory ofProteomics and Analytical Technologies, Science Applications InternationalCorporation-Frederick, Inc., National Cancer Institute, Frederick, Maryland;3Expression Pathology Incorporated, Gaithersburg, Maryland; 4Department ofPharmacology and Physiology,The GeorgeWashington University Medical Center,Washington, District of Columbia; and 5Pathogen Functional Genomics ResourceCenter, J. CraigVenter Institute, Rockville, MarylandReceived 6/8/07; revised10/19/07; accepted12/5/07.Grant support: Intramural Program, National Institute of Dental and CraniofacialResearch and National Cancer Institute, NIH, under contract NO1-CO-12400.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).The content of this publication does not necessarily reflect the views or policies ofthe Department of Health and Human Services, nor does mention of trade names,commercial products, or organization imply endorsement by the U.S. Government.Current address for B.L. Hood and T.P. Conrads: Clinical Proteomics Facility,University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.Requests for reprints: J. Silvio Gutkind, Oral and Pharyngeal Cancer Branch,National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive,Building 30, Room 211, Bethesda, MD 20892-4330. Phone: 301-496-6259;Fax: 301-402-0823; E-mail: [email protected].

F2008 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-1497

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Furthermore, the combination of proteomic analysis with lasercapture microdissection may afford performing the proteomiccharacterization of normal and pathologic cell populations fromclinical specimens, thus reflecting their protein make up as theyexist in vivo (9). We have recently conducted the first proteome-wide analysis of microdissected frozen HNSCC tissue (10).Although protein recovery was acceptable, challenges related tothe poor histology and limited availability of appropriatelypreserved frozen tissue samples still remain.Formalin fixation and tissue embedding in paraffin wax

(FFPE) is a universal approach for tissue processing, histologicevaluation, and routine diagnosis, as it preserves the cellularmorphology and tissue architecture. FFPE clinical specimens,however, are not routinely used for MS-based proteomic studiesbecause formaldehyde-induced cross-linking renders proteinsrelatively insoluble and unsuitable for extraction and subse-quent MS analysis (11). In this study, we combined the use oflaser capture microdissection, newly developed proceduresenabling the extraction of peptides directly from FFPE samples,and optimized chromatographic approaches to undertake alarge-scale proteomic study to identify proteins expressed inFFPE HNSCC tissues. We show that this novel proteomicsplatform enables the identification of hundreds of proteinsexpressed in normal oral epithelium and cancerous HNSCClesions. A large number of differentiation markers, stem cellproteins, and molecules that are likely to play key roles inaberrant cell growth, including proteins involved in cell cyclecontrol, angiogenesis, and metastasis, were identified. Theseproteins may represent novel biomarkers with diagnostic andprognostic value for HNSCC, as well as new potential moleculartargets for pharmacologic intervention in this disease.

Materials andMethods

Tissues. Appropriate paraffin blocks of formalin-fixed tissues fromnormal squamous epithelium (n = 4), and well differentiated (WD;n = 4), moderately differentiated (MD; n = 4), and poorly differentiated

(PD; n = 4) HNSCC were retrieved from the Head and Neck CancerTissue Array Repository (TMA)6 (12), under an approved ResearchActivity Involving Human Subjects protocol, from the Office of HumanSubjects Research, NIH, involving the use of anonymous normal andHNSCC tissues. Before analysis, H&E-stained section from each samplewas evaluated and the suitability of inclusion for the study wasdetermined. Five-micrometer sections were used for all subsequentanalysis.

Immunohistochemistry. Primary antibodies used for validationstudies include mouse anti–desmoglein-3 (Invitrogen; 1:25), rabbitanti-cytokeratin 4 (Abcam; 1:100), mouse anti-cytokeratin 16 (Abcam;1:25), rabbit anti-desmoplakin antibody (Abcam; 1:200), and mouseanti-vimentin (Dako; 1:100). Secondary antibodies conjugated withbiotin (Vector) were used, diluted to 1:400. Tissue slides containingarchival FFPE sections, or tissue microarrays (TMA)6 consisting of 508HNSCC and controls, were processed for immunohistochemicalanalysis as described in Supplementary Material.

Laser capture microdissection and protein extraction. FFPE oralcancer tissue sections were deparaffinized in SafeClear II, hydrated,and stained in Mayer’s hematoxylin followed by dehydration asdescribed in Supplementary Material. For laser capture microdissection,stained uncovered slides were air dried and f20,000 cells werecaptured onto CapSure LCM Caps (MDS Analytical Technologies) usinga PIXCELL IIe microdissection equipment (MDS Analytical Technolo-gies). Caps were transferred to a 0.5 mL sterile Eppendorf tube forprotein extraction using the Liquid Tissue MS Protein Prep kit accordingto the manufacturer’s protocol (Expression Pathology, Inc.), asdescribed in Supplementary Material.

Tandem MS and bioinformatic analysis. FFPE-extracted samples wereprocessed, quantified, and used for nanoflow reversed-phase liquidchromatography followed by tandem MS, as described in Supplemen-tary Material. The spectra were searched against the UniProt humanprotein database (06/2005 release) from the European BioinformaticsInstitute7 using SEQUEST (Thermo Electron). Results were furtherfiltered using software developed in-house to determine uniquepeptides and proteins, which has a predicted error <1.5% (13).

Table 1. Clinical features of HNSCC cases and summary of the liquid chromatography–tandem MS data

Sample Pathology Sex Age Location Total peptides Unique proteins Total (n = 4)

2N N M 71 Oral 1,143 2078N N U 50 Oral 775 14036N N M 63 Oral 620 13737N N M 55 Oral 339 106 39112A WD SCC F 62 Oral 1,454 37614A WD SCC F U Oral 1,570 35615A WD SCC F 60 Oral 1,586 35116A WD SCC F 42 Oral 1,721 323 8661A MD SCC F 67 Oral 1,833 3662A MD SCC M 56 Oral 1,422 3226A MD SCC M U Oral 838 2169A MD SCC M 33 Oral 795 194 72910A PD SCC F 52 Oral 1,202 23911A PD SCC M 72 Oral 1,411 29613A PD SCC U 50 Oral 1,073 25718A PD SCC U U Oral 774 185 676

NOTE: All clinical samples were retrieved from the Head and Neck Tissue Microarray initiative, and chosen based primarily on location within theoral cavity. Samples were assessed for the presence of normal and malignant squamous epithelia that were either WD, MD, or PD. Whereavailable, information on patient gender and age is included. Summary of number of peptides and proteins detected in each sample is included.Abbreviations: N, normal; M, male; F, female; U, unknown.

6 http://www.nidcr.nih.gov/Research/ResearchResources/HeadandNeckTissueArrayInitiative.htm7 http://www.ebi.ac.uk/integr8

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To facilitate the biological interpretation of the extensive protein listsgenerated in these studies, the protein accession numbers were used toclassify the proteins in Gene Ontology categories, based on theirbiological process and molecular and cellular functions (14), and toperform Expression Analysis Systematic Explorer analysis,8 whichenables the discovery of enriched biological themes within gene/protein lists, and the generation of protein annotation tables.

Results

HNSCC samples and laser capture microdissection. To gaininsight into the nature of proteins expressed during HNSCCprogression, we conducted a proteomic analysis of FFPEHNSCCtumors arising within the oral cavity. Tissue samples wereclassified using light microscopic examination of H&E-stainedsections into normal (i.e., oral stratified epithelia lacking

malignant features) or squamous carcinomas that were WD,MD, or PD. Each group consisted of four independent samples.The available clinical information is included in Table 1. Theanalytic workflow for the overall study is depicted in Fig. 1,which is the result of combining two different technologyplatforms, laser capture microdissection, and tandem MS. Asshown schematically (Fig. 1A) and in detail (Fig. 1B), lasercapture microdissection is well suited for the rapid procure-ment of specific cell populations, which are captured onto capsfor immediate processing and analysis. The complexity ofthe resulting peptide mixture extracted from each sample isexemplified in Fig. 1A by the base peak spectrum of a repre-sentative WD HNSCC tumor (right). A tandem MS spectrumidentifying a peptide originating from vimentin is also shown inthis figure. This proteomic platform results in broad dynamicrange of peptide measurements, which may aid in theidentification of important molecules involved in squamouscarcinogenesis as well as biomarkers for HNSCC progression.8 http://david.abcc.ncifcrf.gov

Fig. 1. Work flow for protein analysis ofFFPE oral squamous cell carcinoma.A, different steps involved in the process ofprotein analysis from laser-captured FFPEcancer tissues, including (from left to right)tissue biopsy, laser capture microdissection,sample preparation, and analysis byreversed-phase liquid chromatography ^tandemMS.The latter involves the initialseparation of complex peptide mixturesby Nanoflow reversed-phase liquidchromatography, followed by LITMS. Acomplex base peak chromatogram of arepresentative HNSCC case is included, aswell as the tandemMS spectra of a selectedpeptide whose identity was confirmed asvimentin.B, laser capturemicrodissectionofaWDFFPE oral squamous cell carcinoma(top left).The area of interest is pulsed withlaser (bottom left) and captured cells wereretrieved on a cap (bottom right). Remnanttissue remains on the slide (top right).

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Protein abundance in normal and tumor HNSCC. Samplescontaining f20,000 cells were processed as described inMaterials and Methods and analyzed using gas phase fraction-ation in the m/z dimension (GPFm/z). To enable the compar-ison of protein expression across all tissue samples, we used aspectral count method in which each protein that wasidentified in any one set was evaluated based on the numberof unique peptides identified, and the total number of timesthat those peptides were observed in five successive runs foreach set (Table 1). The number of proteins identified for eachset as a group (all four sample results pooled together) rangedfrom 391 in the normal oral epithelia to 866 total proteins inthe WD HNSCC tumors.The data sets for each group of clinical samples were sorted

based on the number of peptides identified in all tissuescombined and the percentage of these total peptides detected innormal and each of the classified HNSCC tissues. Thoseproteins detected in normal and all HNSCC tissues are listedin Table 2 and sorted based on the number of identifiedpeptides. As indicated, 115 proteins were identified as sharedacross all normal and tumor tissues. The utility of this overallapproach is reflected in the identification of 49 peptides forglyceraldehyde-3-phosphate dehydrogenase, a protein productof a housekeeping gene routinely used to normalize geneexpression experiments. In spite of observing fewer overallpeptides in normal oral epithelia, which may have resulted froma reduced protein recovery from these samples, the relativedetection level of glyceraldehyde-3-phosphate dehydrogenasewas nearly equal across all tissue groups analyzed in this study.Thus, it is also possible that normal tissues might exhibit agreater diversity of molecules, therefore fewer achieving thelowest limit of detection of our current analytic method.Two groups of proteins, cytokeratins and desmosomal

proteins, stood out as the most abundant in all four-tissuesets. With few notable exceptions, most keratins were nearlyequally represented across all tissue samples. For example,cytokeratin 5, a keratin expressed in basal layer of normalstratified epithelia, was the most abundant and was identifiedby a similar number of peptides in each tissue group.Cytokeratin 14, another basal keratin, was less abundant innormal tissues. Conversely, cytokeratin 4, which is expressed inthe nonkeratinazing layer of stratified epithelium, was moreabundant in the normal samples. Desmosomal proteins,including desmoplakin, plakophilin 1, periplakin, and desmo-glein precursors, were prominent in all samples. Also notable inthis list were the family of calcium-binding proteins (calgranu-lin A, calgranulin B, S100 A14, calmodulin-like protein),keratinocyte differentiation markers (involucrin, small pro-line-rich protein 3, profilin 1, cornifin A, cornifin B), and manymembrane-related molecules (Annexin A1, actin-like protein2). A number of proteins including heat shock protein 27(HSP27) (15), HSP70 (16), and glutathione S-transferase (15),expected to be of higher abundance in tumor samples, wereidentified. A number of signaling molecules (e.g., Ras GTPase-activating-like protein IQGAP1, obscurin, tyrosine-proteinkinase ITK) were also identified. Conversely, the DNA excisionrepair protein ERCC-5 was found to be less abundant in tumorsamples. These data indicate that the analysis of FFPE tissuesenables the identification of a broad range of functionallydiverse proteins in normal and tumor squamous epithelia,albeit many exhibit a distinct expression profile.

Proteins detected only in HNSCC. Having identified com-mon proteins in both normal and tumor samples, a list ofmolecules only identified in tumor samples was collated(Table 3). Forty-two proteins unique to HNSCC were readilydetected by multiple peptides (>10). Among them, the mostabundant was vimentin, a protein involved in epithelial-mesenchymal transition. A variety of proteins involved in cellmigration, signaling, and proteolysis were also identified.Eighty-five less abundant proteins (i.e., identified by >4peptides but <10 peptides) across all tumor samples werealso detected. This group included proteins involved in DNAsynthesis, metabolism, and cell signaling. These data providea list of proteins that may play a putative role in tumorprogression.

Proteins detected in normal oral squamous epithelium. Togain insight into the proteins identified exclusively in normalsamples, the initial data set was sorted to filter out moleculesnot found within the tumors, in descending order based onpeptide numbers (Supplementary Table S1). The proteins inthis list were identified by fewer peptides, reflecting their lowerabundance in normal oral squamous epithelium. Interestingly,proteins identified by z3 unique peptides included the low-density lipoprotein receptor-related protein 12 precursor, alsoknown as suppressor of tumorigenicity protein 7, and twoparticular proteins identified with a single peptide included,activin h B chain and adenomatous polyposis coli protein. Thedata indicate that a subset of proteins are expressed preferen-tially in normal tissues, indicating that they could play a role inany of the biological functions done by normal stratified oralepithelium, including maintenance of normal differentiationprogram and tumor suppression.

Proteins detected in differentiated tumor tissues. Proteins ofinterest detected exclusively in the WD tumors, albeit with fewpeptides, included those involved in the dynamic function of thecytoskeleton, as well as molecules stimulating the Notchpathway, such as Delta 4 and Delta 1 (Supplementary TableS2). Proteins of interest and detected only in the MD group withone peptide include Wilms’ tumor-associated protein and eso-phageal cancer–related gene-coding leucine-zipper motif, andunusual cadherins and desmosomal proteins for epithelial cellssuch as placental cadherin and protocadherin g A6 (Supplemen-tary Table S3). Interesting proteins detected only the PD groupinclude the potential oncoprotein AF1q (17), two peptidesderived from epithelial protein lost in neoplasm, and numerousproteins involved in cell cycle control and fatty acid metabolismand membrane trafficking (Supplementary Table S4).

Comparisons of proteins between samples. The list of proteinsidentified in each group was also compared to explore whetherthey could increase our understanding of HNSCC pathogenesisand its progression. Proteins identified only in normal and WDtumors by z3 peptides included the stem cell protein PIWIL3(ref. 18; Table 4; Supplementary Table S5). A number ofproteins were common to WD and MD tumors, represented byoncoprotein DJ-1 (Supplementary Table S6). Similarly, com-mon proteins between MD and PD tumors included the tumorpromigratory protein JWA. Notably, low-abundance proteinsshared by the same group of HNSCC tumors also includedsignal transducer and activator of transcription 3, its activatingkinase, Janus-activated kinase 2, and the key translational regu-lating protein mammalian target of rapamycin (Supplemen-tary Table S7), which are implicated in HNSCC progression

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Table 2. Proteins identified in both normal and tumor HNSCC tissues

Accession Protein Peptides(number)

Normal(%)

WD(%)

MD(%)

PD(%)

P13647 Keratin, type II cytoskeletal 5 (cytokeratin 5; K5) 681 21.73 31.42 23.64 23.20P02533 Keratin, type I cytoskeletal 14 (cytokeratin 14; K14) 449 7.80 42.54 29.18 20.49P08779 Keratin, type I cytoskeletal 16 (cytokeratin 16; K16) 387 8.27 41.09 30.23 20.41P15924 Desmoplakin (DP; 250/210 kDa paraneoplastic pemphigus antigen) 385 15.32 43.90 18.44 22.34P19013 Keratin, type II cytoskeletal 4 (cytokeratin 4; K4) 179 77.65 3.91 12.29 6.15P04264 Keratin, type II cytoskeletal 1 (cytokeratin 1; K1) 146 18.49 22.60 28.77 30.14P14618 Pyruvate kinase, isozymes M1/M2 (CTHBP; THBP1) 136 13.24 40.44 25.74 20.59P04083 Annexin A1 (Annexin I; lipocortin I; calpactin II) 120 21.67 35.00 20.83 22.50Q13835 Plakophilin 1 (band-6-protein, B6P) 95 16.84 30.53 21.05 31.58P06702 Calgranulin B (S100 calcium-binding protein A9) 87 24.14 37.93 19.54 18.39P04792 Heat-shock protein h-1 (HspB1; heat shock 27 kDa protein; HSP 27) 83 4.82 28.92 31.33 34.94P37802 Transgelin-2 (SM22-a homologue) 77 9.09 25.97 33.77 31.17Q04695 Keratin, type I cytoskeletal 17 (cytokeratin 17; K17) 76 6.58 40.79 31.58 21.05P60174 Triosephosphate isomerase (EC 5.3.1.1; TIM; triose-phosphate isomerase) 76 7.89 35.53 22.37 34.21P12111 Collagen a 3(VI) chain precursor 69 2.90 31.88 46.38 18.84P06576 ATP synthase h chain, mitochondrial precursor 67 5.97 32.84 32.84 28.36P32926 Desmoglein 3 precursor (130 kDa pemphigus vulgaris antigen; PVA) 65 15.38 29.23 30.77 24.62P19971 Thymidine phosphorylase precursor (PD-ECGF) 58 1.72 50.00 34.48 13.79P07476 Involucrin 57 8.77 56.14 26.32 8.77P19012 Keratin, type I cytoskeletal 15 (cytokeratin 15; K15) 54 37.04 9.26 18.52 35.19P02545 Lamin A/C (70 kDa lamin) 49 4.08 34.69 32.65 28.57P04406 Glyceraldehyde-3-phosphate dehydrogenase, liver (GAPDH) 49 22.45 24.49 20.41 32.65Q9UBC9 Small proline-rich protein 3 (cornifin h; esophagin; 22 kDa pancornulin) 49 61.22 4.08 6.12 28.57P61978 Heterogeneous nuclear ribonucleoprotein K (hnRNP K) 48 10.42 29.17 27.08 33.33P35527 Keratin, type I cytoskeletal 9 (cytokeratin 9; K9) 46 21.74 23.91 8.70 45.65P62263 40S ribosomal protein S14 (PRO2640) 44 2.27 29.55 45.45 22.73P27482 Calmodulin-related protein NB-1 (calmodulin-like protein; CLP) 43 6.98 41.86 30.23 20.93P10809 60 kDa heat shock protein, mitochondrial precursor (Hsp60) 42 14.29 33.33 11.90 40.48P35908 Keratin, type II cytoskeletal 2 epidermal (cytokeratin 2e; K2e) 39 30.77 17.95 2.56 48.72P02462 Collagen a 1(IV) chain precursor 38 34.21 21.05 26.32 18.42P55072 Transitional endoplasmic reticulum ATPase (TER ATPase) 37 2.70 51.35 32.43 13.51P30101 Protein disulfide-isomerase A3 precursor (disulfide isomerase ER-60; ERp60) 36 2.78 36.11 38.89 22.22P11021 78 kDa glucose-regulated protein precursor (GRP 78) 36 2.78 47.22 27.78 22.22P35579 Myosin heavy chain, nonmuscle type A (cellular myosin heavy chain, type A) 36 2.78 41.67 30.56 25.00Q9Y446 Plakophilin3 35 28.57 28.57 31.43 11.43Q01469 Fatty acid-binding protein, epidermal (E-FABP; PA-FABP) 34 17.65 32.35 29.41 20.59Q09666 Neuroblast differentiation associated protein AHNAK (desmoyokin) 31 16.13 32.26 35.48 16.13P22626 Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/hnRNP B1) 30 6.67 36.67 23.33 33.33P05387 60S acidic ribosomal protein P2 29 24.14 24.14 27.59 24.14Q14679 Tubulin tyrosine ligase-like protein 4 27 11.11 25.93 40.74 22.22Q9Y2L5 TRS85 homologue 26 7.69 34.62 30.77 26.92P61160 Actin-like protein 2 (actin-related protein 2) 26 7.69 38.46 30.77 23.08P62917 60S ribosomal protein L8 26 19.23 26.92 26.92 26.92Q14134 Tripartite motif protein 29 (ataxia-telangiectasia group D-associated protein) 26 23.08 34.62 19.23 23.08P12821 Angiotensin-converting enzyme, somatic isoform precursor (ACE) 25 60.00 8.00 4.00 28.00P47929 Galectin-7 (Gal-7; HKL-14; PI7; p53-induced protein 1) 24 8.33 66.67 12.50 12.50P07737 Profilin-1 (profilin I) 24 16.67 20.83 29.17 33.33P36578 60S ribosomal protein L4 (L1) 23 26.09 8.70 17.39 47.83P09211 Glutathione S-transferase P (GST class-pi; GSTP1-1) 22 4.55 31.82 22.73 40.91P52272 Heterogeneous nuclear ribonucleoprotein M (hnRNP M) 22 9.09 18.18 22.73 50.00P07195 L-lactate dehydrogenase B chain (LDH heart subunit; LDH-H) 22 13.64 36.36 13.64 36.36P48643 T-complex protein 1, epsilon subunit (TCP-1e; CCT-e) 21 4.76 42.86 28.57 23.81P10599 Thioredoxin (ATL-derived factor; ADF) 21 14.29 28.57 38.10 19.05P06748 Nucleophosmin (NPM; nucleolar phosphoprotein B23) 21 14.29 28.57 14.29 42.86P11142 Heat shock cognate 71 kDa protein (heat shock 70 kDa protein 8) 20 5.00 30.00 25.00 40.00P49411 Elongation factor Tu, mitochondrial precursor (EF-Tu; P43) 20 10.00 30.00 25.00 35.00P14625 Endoplasmin precursor (94 kDa glucose-regulated protein; GRP94) 20 15.00 45.00 20.00 20.00P05109 Calgranulin A (migration inhibitory factor–related protein 8; MRP-8) 20 35.00 35.00 15.00 15.00Q6ZUU7 Hypothetical protein FLJ43318 19 26.32 15.79 42.11 15.79Q9HCY8 S100 calcium-binding protein A14 (S114) 19 36.84 21.05 26.32 15.79P30044 Peroxiredoxin 5, mitochondrial precursor (Prx-V) 18 5.56 16.67 44.44 33.33Q14764 Major vault protein (MVP; lung resistance-related protein) 18 5.56 33.33 38.89 22.22P18206 Vinculin (metavinculin) 18 11.11 50.00 27.78 11.11Q14574 Desmocollin 3 precursor (desmocollin 4; HT-CP) 18 16.67 22.22 22.22 38.89P46781 40S ribosomal protein S9 18 16.67 33.33 16.67 33.33

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(19, 20). Full lists of proteins for these comparisons areavailable in Supplementary Tables S5 to S7.Gene ontology analysis. Analysis of each sample set resulted

in the identification of a large number of proteins. Therefore, itwas necessary to integrate these data based on our currentlyavailable knowledge of biological functions to determine theirindividual biological roles and to recognize categories ofproteins that may be underrepresented or overrepresented intumors. For this, we used bioinformatic tools to classify

proteins across each of the HNSCC tumor types into differentgene ontology categories. This approach enabled the examina-tion of the overall cellular compartment in which the identifiedproteins are expected to reside and function. Although aminimal difference was observed between normal and HNSCCsamples, we found that 133-295, 10-21, 77-185, and 86-184proteins were associated with the cytoplasm, extracellularmatrix, membrane, and the nucleus, respectively. Analysis ofthe biological processes in which these proteins act revealed

Table 2. Proteins identified in both normal and tumor HNSCC tissues (Cont’d)

Accession Protein Peptides(number)

Normal(%)

WD(%)

MD(%)

PD(%)

P62913 60S ribosomal protein L11 (CLL-associated antigen KW-12) 17 5.88 47.06 23.53 23.53P62899 60S ribosomal protein L31 16 6.25 43.75 18.75 31.25Q99456 Keratin, type I cytoskeletal 12 (cytokeratin 12; K12; CK 12) 14 7.14 57.14 21.43 14.29Q02413 Desmoglein 1 precursor (desmosomal glycoprotein 1; DG1; DGI) 14 14.29 35.71 42.86 7.14P83731 60S ribosomal protein L24 (ribosomal protein L30) 13 7.69 38.46 23.08 30.77P46940 Ras GTPase-activating-like protein IQGAP1 (p195) 13 7.69 30.77 30.77 30.77P00505 Aspartate aminotransferase, mitochondrial precursor (transaminase A) 13 15.38 23.08 7.69 53.85P78371 T-complex protein 1, h subunit (TCP-1-h; CCT-h) 13 15.38 30.77 30.77 23.08P50990 T-complex protein 1, u subunit (TCP-1-u; CCT-u) 13 23.08 38.46 15.38 23.08O60506 Heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) 12 8.33 41.67 41.67 8.33Q00610 Clathrin heavy chain 1 (CLH-17) 12 16.67 33.33 8.33 41.67O60437 Periplakin (195 kDa cornified envelope precursor protein) 12 25.00 25.00 25.00 25.00Q07020 60S ribosomal protein L18 12 33.33 33.33 25.00 8.33P30041 Peroxiredoxin 6 (antioxidant protein 2) 11 18.18 18.18 27.27 36.36Q96AA2 Obscurin 11 18.18 36.36 27.27 18.18Q5U4P6 KHSRP protein 11 18.18 45.45 9.09 27.27Q08881 Tyrosine-protein kinase ITK/TSK 11 18.18 36.36 27.27 18.18P13639 Elongation factor 2 (EF-2) 11 27.27 45.45 9.09 18.18P35321 Cornifin A (small proline-rich protein IA; SPR-IA; SPRK) 11 27.27 45.45 9.09 18.18P22528 Cornifin B (small proline-rich protein IB; SPR-IB) 11 36.36 36.36 9.09 18.18Q01518 Adenylyl cyclase-associated protein 1 (CAP 1) 10 10.00 30.00 20.00 40.00P08572 Collagen a 2(IV) chain precursor 10 10.00 50.00 20.00 20.00Q96MG1 Hypothetical protein FLJ32421 10 10.00 50.00 30.00 10.00O75312 Zinc-finger protein ZPR1 (zinc finger protein 259) 10 30.00 10.00 10.00 50.00Q8TBA0 Chromosome 8 open reading frame 21 10 30.00 20.00 10.00 40.00Q96CN5 Hypothetical protein MGC20806 10 30.00 40.00 10.00 20.00P40121 Macrophage capping protein (actin-regulatory protein CAP-G) 9 11.11 11.11 44.44 33.33P06733 a Enolase (Enolase 1) 9 11.11 22.22 33.33 33.33P12035 Keratin, type II cytoskeletal 3 (cytokeratin 3; K3) 9 11.11 44.44 22.22 22.22O00299 Chloride intracellular channel protein 1 (nuclear chloride ion channel 27) 9 11.11 44.44 22.22 22.22Q02388 Collagen a 1(VII) chain precursor (long-chain collagen; LC collagen) 9 22.22 11.11 11.11 55.56Q9Y6X9 Zinc finger CW-type coiled-coil domain protein 1 9 22.22 55.56 11.11 11.11P04080 Cystatin B (liver thiol proteinase inhibitor; CPI-B; Stefin B) 9 33.33 33.33 22.22 11.11P49368 T-complex protein 1, g subunit (TCP-1-g; CCT-g) 8 12.50 12.50 25.00 50.00Q6ZT17 Hypothetical protein FLJ45043 8 12.50 25.00 25.00 37.50O15353 Forkhead box protein N1 (transcription factor winged-helix nude) 8 37.50 25.00 25.00 12.50P28715 DNA excision repair protein ERCC-5 8 50.00 25.00 12.50 12.50P24534 Elongation factor 1-h (EF-1-h) 7 14.29 14.29 28.57 42.86Q8N7I6 Hypothetical protein FLJ25506 6 16.67 16.67 33.33 33.33P62277 40S ribosomal protein S13 6 16.67 16.67 16.67 50.00Q99551 Transcription termination factor, mitochondrial precursor (mTERF) 6 16.67 33.33 33.33 16.67P31153 S-adenosylmethionine synthetase g form (AdoMet synthetase) 6 16.67 50.00 16.67 16.67Q9UKG9 Peroxisomal carnitine O-octanoyltransferase (COT) 6 33.33 16.67 33.33 16.67Q15369 Transcription elongation factor B polypeptide 1 (elongin C) 6 50.00 16.67 16.67 16.67Q06830 Peroxiredoxin 1 (EC 1.11.1.-; thioredoxin peroxidase 2) 5 20.00 20.00 40.00 20.00O75151 PHD finger protein 2 (GRC5) 4 25.00 25.00 25.00 25.00O60231 Putative pre-mRNA splicing factor RNA helicase (DEAH-box protein 16) 4 25.00 25.00 25.00 25.00P52907 F-actin capping protein a-1 subunit (CapZ a-1) 4 25.00 25.00 25.00 25.00Q99698 Lysosomal trafficking regulator (Beige homologue) 4 25.00 25.00 25.00 25.00Q8TBU6 Hypothetical protein FLJ11848 4 25.00 25.00 25.00 25.00

NOTE: Proteins that were identified as common in all normal and HNSCC tissues were sorted across data sets of the different tissue samples asdescribed in Materials and Methods, based on the corresponding peptide number in descending order and their relative distribution (%) acrossthe different samples: normal, WD, MD, and PD HNSCC. Accession number for each protein is also indicated.

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Table 3. Proteins identified only in tumoral HNSCC tissues

Accession Protein Peptides(number)

WD(%)

MD(%)

PD(%)

P08670 Vimentin 79 36.71 44.30 18.99P02452 Collagen a 1(I) chain precursor 57 43.86 29.82 26.32P23396 40S ribosomal protein S3 27 59.26 29.63 11.11Q15365 Poly(rC)-binding protein 1 (a-CP1; hnRNP-E1; nucleic acid binding protein SUB2.3) 25 36.00 36.00 28.00P23246 Splicing factor, proline-and glutamine-rich (PSF) 23 34.78 21.74 43.48P06396 Gelsolin precursor (actin-depolymerizing factor; ADF; brevin; AGEL) 23 47.83 30.43 21.74P67936 Tropomyosin a 4 chain (tropomyosin 4; TM30p1) 22 40.91 31.82 27.27Q8NFW1 a1 Type XXII collagen 22 31.82 40.91 27.27P29401 Transketolase (EC 2.2.1.1; TK) 20 50.00 35.00 15.00Q8NC51 Plasminogen activator inhibitor 1 (PAI1) 19 42.11 42.11 15.79P07108 Acyl-CoA-binding protein (ACBP; diazepam binding inhibitor, DBI; endozepine, EP) 19 42.11 31.58 26.32Q96AE4 Far upstream element binding protein 1 (FUSE binding protein 1, FBP) 18 27.78 27.78 44.44P13796 L-plastin (lymphocyte cytosolic protein 1, LCP-1; LC64P) 17 35.29 35.29 29.41P22105 Tenascin X precursor (TN-X; hexabrachion-like) 17 41.18 29.41 29.41O94851 Protein MICAL-2 17 23.53 47.06 29.41O43707 a-actinin 4 (Nonmuscle a-actinin 4; F-actin cross-linking protein) 16 43.75 18.75 37.50P26038 Moesin (membrane-organizing extension spike protein) 15 13.33 60.00 26.67Q9HDC5 Junctophilin 1 (junctophilin type 1, JP-1) 15 20.00 33.33 46.67P31946 14-3-3 Protein h/a (protein kinase C inhibitor protein-1; KCIP-1; protein 1054) 15 33.33 40.00 26.67P28065 Proteasome subunit h type 9 precursor (proteasome chain 7; macropain chain 7) 15 46.67 40.00 13.33Q8N7Z2 Hypothetical protein FLJ40198 15 40.00 33.33 26.67P07237 Protein disulfide-isomerase precursor (PDI) 15 46.67 26.67 26.67P00354 Glyceraldehyde-3-phosphate dehydrogenase, muscle (EC 1.2.1.12; GAPDH) 14 42.86 28.57 28.57Q9P2E9 Ribosome-binding protein 1 (ribosome receptor protein) 14 42.86 35.71 21.43P02461 Collagen a 1(III) chain precursor 14 28.57 42.86 28.57P42224 Signal transducer and activator of transcription 1-a/h 14 57.14 28.57 14.29Q9BVC6 Hypothetical protein MGC5508 13 23.08 30.77 46.15P05783 Keratin, type I cytoskeletal 18 (cytokeratin 18; K18; CK 18) 13 23.08 15.38 61.54P22314 Ubiquitin-activating enzyme E1 (A1S9 protein) 13 53.85 23.08 23.08P07339 Cathepsin D precursor (EC 3.4.23.5) 13 61.54 23.08 15.38P00338 L-Lactate dehydrogenase A chain (EC 1.1.1.27; LDH-A; LDH muscle subunit; LDH-M) 13 69.23 15.38 15.38Q99623 B-cell receptor-associated protein BAP37 (D-prohibitin) 12 33.33 16.67 50.00P06703 Calcyclin (prolactin receptor associated protein, PRA) 12 66.67 25.00 8.33P11166 Solute carrier family 2, facilitated glucose transporter, member 1 11 27.27 27.27 45.45P31146 Coronin-1A (coronin-like protein p57; coronin-like protein A) 11 27.27 45.45 27.27P04179 Superoxide dismutase [Mn], mitochondrial precursor (EC 1.15.1.1) 10 20.00 30.00 50.00O15083 ERC protein 2 10 30.00 30.00 40.00Q9NZM4 Glioma tumor suppressor candidate region gene 1 protein 10 30.00 30.00 40.00P52565 Rho GDP-dissociation inhibitor 1 (Rho GDI 1; Rho-GDI a) 10 30.00 50.00 20.00P62851 40S ribosomal protein S25 10 40.00 40.00 20.00Q9BPX3 Condensin subunit 3 (chromosome-associated protein G; NY-MEL-3 antigen) 10 50.00 20.00 30.00P20908 Collagen a 1(V) chain precursor 10 50.00 30.00 20.00Q9Y6R0 Numb-like protein (Numb-R) 9 22.22 44.44 33.33P55786 Puromycin-sensitive aminopeptidase (EC 3.4.11.-; PSA) 9 33.33 22.22 44.44P26641 Elongation factor 1-g (EF-1-g; eEF-1B g; PRO1608) 9 44.44 11.11 44.44P40926 Malate dehydrogenase, mitochondrial precursor (EC 1.1.1.37) 9 44.44 22.22 33.33P20700 Lamin B1 9 44.44 33.33 22.22Q8WY91 THAP domain protein 4 (PP238; CGI-36) 8 25.00 25.00 50.00Q9NZT1 Calmodulin-like protein 5 (calmodulin-like skin protein) 8 50.00 25.00 25.00P08195 4F2 cell-surface antigen heavy chain (4F2hc; CD98 antigen) 8 37.50 37.50 25.00P27797 Calreticulin precursor (CRP55; calregulin; HACBP; ERp60; grp60) 8 50.00 12.50 37.50P23368 NAD-dependent malic enzyme, mitochondrial precursor (malic enzyme 2) 8 62.50 12.50 25.00P27348 14-3-3 protein H (14-3-3 protein u; 14-3-3 protein T-cell; HS1 protein) 7 28.57 42.86 28.57P14314 Glucosidase II h subunit precursor (PKCSH) 7 28.57 57.14 14.29Q02487 Desmocollin 2A/2B precursor (desmosomal glycoprotein II and III; desmocollin-3) 7 42.86 28.57 28.57Q01105 SET protein (phosphatase 2A inhibitor I2PP2A; I-2PP2A; template activating factor I) 7 42.86 42.86 14.29P05023 Sodium/potassium-transporting ATPase a-1 chain precursor (sodium pump 1) 7 28.57 42.86 28.57P32119 Peroxiredoxin 2 (thioredoxin peroxidase 1; natural killer cell enhancing factor B) 7 42.86 28.57 28.57Q6ZRH9 Hypothetical protein FLJ46347 7 42.86 42.86 14.29P27824 Calnexin precursor (MHC class I antigen-binding protein p88) 7 42.86 14.29 42.86Q9Y2G4 Ankyrin repeat domain protein 6 7 42.86 28.57 28.57Q9P2H1 KIAA1376 protein (fragment) 7 42.86 42.86 14.29Q6UVZ0 GLSR2492 7 57.14 28.57 14.29Q96LT0 Hypothetical protein FLJ25091 7 57.14 14.29 28.57P18827 Syndecan-1 precursor (SYND1; CD138 antigen) 7 71.43 14.29 14.29

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several groups of molecules that were highly represented intumors, many involved in cell adhesion, cell cycle, celldifferentiation, cellular metabolic process, cell motility, endo-peptidase activity, signal transduction, and gene transcriptionand translation, closely aligned with the processes of carcino-genesis. Indeed, a large number of proteins belonging to thesefunctional groups were ascribed to tumors, and to their normaltissue counterpart, albeit to a lesser extent (SupplementaryTable S8). A detailed list of molecules predicted to participate insignal transduction (238 proteins), protein phosphorylation(39 proteins), endopeptidase activity (35 proteins), cellmotility (47 proteins), cell cycle regulation (99 proteins), andcell adhesion (71 proteins) across normal tissues and thedifferent tumor phenotypes is provided in SupplementaryTables S9 to S14.Detection of novel proteins in normal and tumor HNSCC. A

key advantage afforded by this proteome-wide analysis is thepotential for discovery of new molecules yet to be described fornormal and malignant oral squamous tissues. Several hypo-

thetical novel proteins, whose existence is predicated oncomputational analysis of open reading frames (21), werefound to be present in normal and tumor samples. Theseproteins were collated and organized in descending order basedon peptide number. As seen in Supplementary Table S15, themajority of the proteins with z4 peptides were detected ineither tumor or normal samples. Collectively, the emerginginformation identified many novel proteins in normal andtumor samples, whose nature can now be analyzed. In thisregard, structural features of these previously predicted proteinsare quite diverse and suggestive of multiple roles in signaltransduction, cell communication, and secretion, amongothers. Future work could help establish their possible role inHNSCC development and progression.

Proteins identified as of interest for HNSCC. The ability todiscriminate subsets of proteins differentially abundant withinnormal oral squamous epithelia and tumors exhibiting distinctdifferentiation characteristics provided an opportunity to minethe data to identify proteins of interest as well as putative

Table 3. Proteins identified only in tumoral HNSCC tissues (Cont’d)

Accession Protein Peptides(number)

WD(%)

MD(%)

PD(%)

P62829 60S ribosomal protein L23 (ribosomal protein L17) 7 57.14 28.57 14.29P61088 Ubiquitin-conjugating enzyme E2 N (ubiquitin-protein ligase N) 7 42.86 28.57 28.57P84098 60S ribosomal protein L19 7 71.43 14.29 14.29P05164 Myeloperoxidase precursor (EC 1.11.1.7; MPO) 7 71.43 14.29 14.29P62258 14-3-3 protein epsilon (14-3-3E) 6 16.67 66.67 16.67Q6ZVR1 Hypothetical protein FLJ42200 6 16.67 66.67 16.67P37290 IFN y-1 precursor 6 16.67 50.00 33.33Q5VVY7 OTTHUMP00000040479 6 50.00 33.33 16.67O75874 Isocitrate dehydrogenase [NADP] cytoplasmic (oxalosuccinate decarboxylase) 6 33.33 16.67 50.00Q12931 Heat shock protein 75 kDa, mitochondrial precursor (HSP 75; TRAP-1) 6 33.33 33.33 33.33P20794 Serine/threonine-protein kinase MAK (male germ cell-associated kinase) 6 33.33 50.00 16.67Q99797 Mitochondrial intermediate peptidase, mitochondrial precursor (MIP) 6 50.00 16.67 33.33Q96RJ0 Trace amine receptor 1 (TaR-1) 6 66.67 16.67 16.67P61026 Ras-related protein Rab-10 6 50.00 16.67 33.33Q16401 26S proteasome non-ATPase regulatory subunit 5 (26S proteasome subunit S5B) 6 50.00 33.33 16.67Q9NXG7 Hypothetical protein FLJ20261 5 20.00 20.00 60.00Q15717 ELAV-like protein 1 (Hu-antigen R; HuR) 5 40.00 20.00 40.00P35232 Prohibitin 5 20.00 20.00 60.00P12532 Creatine kinase, ubiquitous mitochondrial precursor (U-MtCK) 5 20.00 60.00 20.00Q6IQ21 Hypothetical protein FLJ20582 5 40.00 40.00 20.00Q8N442 Hypothetical protein FLJ13220 5 40.00 40.00 20.00Q96GY3 Protein F25965 5 40.00 20.00 40.00Q13029 PR-domain zinc finger protein 2 (retinoblastoma protein-interacting zinc-finger protein) 5 40.00 20.00 40.00Q13442 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein) 5 40.00 40.00 20.00Q9UHD9 Ubiquilin 2 (protein linking IAP with cytoskeleton-2; hPLIC-2) 5 40.00 40.00 20.00P82914 28S ribosomal protein S15, mitochondrial precursor (S15mt; MRP-S15; DC37) 5 60.00 20.00 20.00O95571 HSCO protein (ETHE1 protein) 5 60.00 20.00 20.00Q9H8N1 Hypothetical protein FLJ13385 4 25.00 25.00 50.00Q6P3R8 MGC75495 protein (OTTHUMP00000040896) 4 25.00 25.00 50.00P16401 Histone H1.5 (histone H1a) 4 25.00 50.00 25.00Q8WXX7 Autism susceptibility gene 2 protein 4 25.00 50.00 25.00Q9UIG0 Bromodomain adjacent to zinc finger domain protein 1B (hWALP2) 4 50.00 25.00 25.00Q86VX5 SLC25A28 protein (fragment) 4 50.00 25.00 25.00Q9UHD8 Septin 9 (MLL septin-like fusion protein; septin D1) 4 50.00 25.00 25.00Q13148 TAR DNA-binding protein-43 (TDP-43) 4 50.00 25.00 25.00Q13724 Mannosyl-oligosaccharide glucosidase 4 50.00 25.00 25.00O00303 Eukaryotic translation initiation factor 3 subunit 5 (eIF-3e) 4 50.00 25.00 25.00P23381 Tryptophanyl-tRNA synthetase (TrpRS; hWRS) 4 50.00 25.00 25.00P13804 Electron transfer flavoprotein a-subunit, mitochondrial precursor (a-ETF) 4 50.00 25.00 25.00

NOTE: Proteins identified exclusively in the tumor samples were sorted as described in Materials and Methods based on their peptide number indescending order and their relative distribution (%) across the different samples: WD, MD, and PD.

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biomarkers of HNSCC development and progression. Aselected group of molecules identified as being differentiallyabundant were sorted by total peptide number and theirrelative distribution across the normal and tumor samples(Table 4). Notably, the peptide distribution of the cytokeratins14, 17, and 16 was observed to be lower in the normal samplescompared with the WD, MD, and PD tumor samples. Bycontrast, the peptide distribution of cytokeratin 4 was higher innormal (f77%) compared with the tumor samples (f4-12%).Also included in the list are cytokeratins 7 and 18 albeit with alower peptide number. The relative distribution indicates thatthese molecules are less abundant in normal samples than inMD and PD tumors, respectively. Molecules involved in cell-to-cell interaction were also identified as potential biomarkers andinclude desmoplakin, democollin 2A/2B, demoglein 3 precur-sor, plakophilin 1, and plakophilin 3. The total peptide numberfor this group of proteins ranged from 7 to 385, with a relative

similar distribution in all samples with the exception ofdemocollin 2A/2B, which was undetectable in normal samples.Furthermore, from the total peptide number of 385 fordesmoplakin, f44% of these were detected in well-differenti-ated tumors and in the other samples indicating close to equallevels. Other proteins of interest include HSP27 and HSP70,vimentin, glutathione S-transferase, and integrin h4. In all theseproteins, the distribution of peptides was low to undetectable innormal samples when compared with the tumors. Among thelatter, we found proteins that may play a role in tumor progres-sion, such as SET protein (phosphatase 2A inhibitor I2PP2A)and ELAV-like protein 1 (Hu-antigen R), and many surfaceproteins, including 4F2 cell surface antigen heavy chain (CD98),and, as described above, integrin h4 (CD104 antigen), whichmay represent promising markers to study tumor progression.Validation of biomarkers by immunohistochemistry. Having

identified proteins of potential interest to HNSCC progression,

Table 4. Representative proteins of interest for HNSCC

Accession Protein Peptides (number) Normal (%) WD (%) MD (%) PD (%)

P02533 Keratin, type I cytoskeletal 14 449 7.80 42.54 29.18 20.49P08779 Keratin, type I cytoskeletal 16 387 8.27 41.09 30.23 20.41P15924 Desmoplakin (DP) 385 15.32 43.90 18.44 22.34P19013 Keratin, type II cytoskeletal 4 179 77.65 3.91 12.29 6.15P04264 Keratin, type II cytoskeletal 1 146 18.49 22.60 28.77 30.14Q13835 Plakophilin 1 (band-6-protein, B6P) 95 16.84 30.53 21.05 31.58P04792 Heat-shock protein h1 (HspB1; HSP 27) 83 4.82 28.92 31.33 34.94P08670 Vimentin 79 0.00 36.71 44.30 18.99Q04695 Keratin, type I cytoskeletal 17 76 6.58 40.79 31.58 21.05P32926 Desmoglein 3 precursor 65 15.38 29.23 30.77 24.62P07476 Involucrin 57 8.77 56.14 26.32 8.77Q9UBC9 Small proline-rich protein 3 (cornifin h) 49 61.22 4.08 6.12 28.57P11021 78 kDa Glucose-regulated protein (GRP 78) 36 2.78 47.22 27.78 22.22Q9Y446 Plakophilin 3 35 28.57 28.57 31.43 11.43P09211 Glutathione S-transferase P (GSTP1-1) 22 4.55 31.82 22.73 40.91P11142 Heat shock cognate 71 kDa protein 20 5.00 30.0 025.00 40.00Q8NC51 Plasminogen activator inhibitor 1 (PAI1) 19 0.00 42.11 42.11 15.79Q14764 Major vault protein (MVP) 18 5.56 33.33 38.89 22.22O43707 a-actinin 4 (nonmuscle a-actinin 4) 16 0.00 43.75 18.75 37.50P42224 Signal transducer and activator of transcription 1 14 0.00 57.14 28.57 14.29Q99456 Keratin, type I cytoskeletal 12 14 7.14 57.14 21.43 14.29P05783 Keratin, type I cytoskeletal 18 13 0.00 23.08 15.38 61.54P46940 Ras GTPase-activating-like protein IQGAP1 (p195) 13 7.69 30.77 30.77 30.77P07339 Cathepsin D precursor 13 0.00 61.54 23.08 15.38P08729 Keratin, type II cytoskeletal 7 11 0.00 0.00 90.90 10.10P52565 Rho GDP-dissociation inhibitor 1 (Rho GDI 1) 10 0.00 30.00 50.00 20.00P12821 PIWIL 3 protein 9 88.00 11.00 0.00 0.00O15353 Forkhead box protein N1 8 37.50 25.00 25.00 12.50P08195 4F2 cell-surface antigen (CD98) 8 0.00 37.50 37.50 25.00P28715 DNA excision repair protein ERCC-5 8 50.00 25.00 12.50 12.50Q02487 Desmocollin 2A/2B precursor (desmocollin-3) 7 0.00 42.86 28.57 28.57O14757 Serine/threonine-protein kinase Chk1 7 57.00 14.00 0.00 29.00Q01105 SET protein (phosphatase 2A inhibitor I2PP2A) 7 0.00 42.86 42.86 14.29Q13015 Protein AF1q 6 0.00 0.00 0.00 100.00Q15717 ELAV-like protein 1 (Hu-antigen R; HuR) 5 0.0 040.00 20.00 40.00P35232 Prohibitin 5 0.00 20.00 20.00 60.00P00533 Epidermal growth factor receptor (EGFR) 4 0.00 50.00 0.00 50.00Q9UHD8 Septin 9 (MLL septin-like fusion protein) 4 0.00 50.00 25.00 25.00P16144 Integrin h4 precursor (CD104) 4 0.00 0.00 25.00 75.00O43157 Plexin B1 precursor (semaphorin receptor SEP) 3 0.00 33.00 33.00 33.00O14511 Pro-neuregulin-2 (pro-NRG2) 3 0.00 33.00 33.00 33.00Q02224 Centromeric protein E (CENP-E protein) 3 0.00 33.00 33.00 33.00

NOTE: Known proteins from the total list that may represent proteins of interest as well as potential biomarkers were sorted based on their totalpeptide number and relative distribution (%) across the normal and WD, MD, and PD tumor samples.

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a subset of these were chosen for validation based on therelative abundance of corresponding peptides in the differentsamples analyzed. Validation studies were conducted usingstandard immunohistochemistry in archival HNSCC tissuesfollowed by in-depth analysis of hundreds of HNSCC casesusing a recently developed HNSCC-specific TMA. As seen inFig. 2A, cytokeratin 4 was detected predominantly in thesuprabasal layers of the control normal tissues, whereas theexpression of this molecule in HNSCC tumors was restrictedto only few cells in <20% of WD tumors. Most MD to WDcells stained positive for cytokeratin 16, whereas in the normaltissues this protein was present in the suprabasal layers of theoral squamous epithelium (second from top). Desmoplakin(third from top) was found to be predominantly membranous,with a higher immunoreactivity in the suprabasal areas of thesquamous epithelium in normal tissues. Tumor cells showedintense staining of the membrane together with a moderate tostrong cytoplasmic staining, distributed mostly along the moredifferentiated areas of all the tumor cases examined. Analysisof desmoglein-3 (fourth from top) showed sharp stainingwithin the membrane in normal oral squamous mucosa witha stronger signal in suprabasal layers. Desmoglein-3 was alsopositive in all tumor samples, following a membranedistribution and a less intense cytoplasmic signal. Immuno-detection of vimentin (bottom) was positive in only a fewnormal isolated cells with very distinct dendritic-like mor-phology in the oral squamous epithelium, whereas theunderlying stromal cells were all intensely stained. By contrast,a high proportion of the malignant squamous cells in thetumor cases showed increased immunoreactivity to vimentin.Collectively, the data indicate that the profile of proteinexpression identified using MS is reflected in an independentarchival HNSCC sample set.The previous data indicated that a subset of proteins

(cytokeratin 4, cytokeratin 16, vimentin, and desmoplakin)could effectively distinguish the differing differentiation com-partments in archival HNSCC tissues. Therefore, we chose touse an oral cancer–specific TMA for the high-throughputstaining and scoring of these predictive immunohistochemicalmarkers in this cancer type. As shown in Fig. 2B (top), althoughcytokeratin 4 was poorly expressed in the majority of thetumors (left and inset), strong staining for cytokeratin 16 inHNSCC was observed almost exclusively in WD tumors orotherwise WD areas (second from left and inset), as most MDand PD tumors failed to react strongly. Vimentin immunore-activity was almost exclusively limited to a subset of malignanttumors, and in the majority of the cases this staining was focal(third from left and inset). Finally, staining for desmoplakin wasstrongly positive in normal tissues as well as in almost all tumorsamples (right and inset).A semiquantitative analysis was applied to the TMA staining.

As indicated in Fig. 2B (bottom), cytokeratin 4 and cytokeratin16 scored positive in normal tissues based on the stainingpattern of the suprabasal layer, and their expression in tumorswas often restricted to WD areas. For the evaluation of vimentin(third from left) and desmoplakin (right), we classified cellstaining in each tissue core as positive and negative because noclear correlation with tumor differentiation was noted from theinitial analysis. Of note, vimentin and desmoplakin wereassessed to be negative and strongly positive, respectively, in allthe relevant normal oral epithelial tissues analyzed.

Discussion

In this study, we describe the utility of a novel proteomicsplatform for the global detection of expressed proteins in FFPEtissues and its use for biomarker discovery and identification ofproteins that may contribute to HNSCC development andprogression. This approach enabled identification of a largenumber of molecules, including cytokeratins and intermediatefilament proteins, differentiation markers, proteins involvedin stem cell maintenance, signal transduction and cell cycleregulation, growth and angiogenic factors, matrix-degradingproteases, and proteins with tumor suppressive and oncogenicpotential. Of interest, detection and relative expression of manyof these molecules followed a distinct pattern in normalsquamous epithelia and WD, MD, and PD HNSCC tumortissues. The ability to correlate protein expression profiles withhistopathologic classification of disease may allow the devel-opment of novel biomarkers of diagnostic and prognostic valueand may help identify novel targets for therapeutic interventionin HNSCC.

Certain advantages embedded within the workflow devel-oped for this study include the efficient solubilization anddigestion of proteins from FFPE archival tissue withoutfractionation, such that they are amenable for identificationusing tandem MS for a complete proteomic representation (7).Furthermore, optimization of combining laser capture micro-dissection with shotgun proteomic technologies enabled thedetection of proteins expressed primarily within the tumor cellsrather than in the stroma and other complex cellularcomponents of the tumor microenvironment. Although theexactness of MS identifications can be challenging, particularlyfor low abundant proteins, current MS instrumentation andimproved bioinformatic capabilities provide a high probabilityof protein identification (22). Thus, rather than reportingproteins identified by two or more unique peptide sequencematches, a common practice in the field that may sacrificemany correct protein identifications derived from high-qualitysingle hits, we chose to report here all proteins identified basedon stringent biophysical and statistical criteria. Indeed, theability to take advantage of new technological developmentsmay now enable the discovery of molecules that althoughpresent in low abundance may nevertheless play importantbiological roles in tumor development.

In this regard, it was encouraging that certain proteinsindicative of ‘‘proof of principle’’ could be readily detected. Forexample, the total peptide number for glyceraldehyde-3-phosphate dehydrogenase, a protein frequently used fornormalization, was nearly equally distributed across thedifferent samples, suggesting that the protein recovery wassimilar for each group of tissues. Further validation of ouranalysis was illustrated by the identification of members of thetaste receptors (T2R13, T2R38), olfactory receptors (5M11,13C4, JCG2), and oral facial proteins (oral-facial-digitalsyndrome 1 protein), each with a single peptide. Althoughthese proteins may not be involved in cancer, they arenevertheless known to be expressed in oral squamous tissues(23–25). The abundance of the cytokeratins (1, 4, 5, 7, 14,16–18) and the desmosomal proteins (desmoplakin, desmo-glein 3, desmocollin, epiplakin, plakophilins) was striking,particularly when considering the limited amount of sampleavailable for the proteomic analysis. Noteworthy, desmoglein 1

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and desmoglein 3 are both expressed in the skin, but the130 kDa molecule desmoglein 3 is preferentially expressed inoral epithelium (26). As these molecules function together tomaintain structural integrity of the normal oral epithelium,changes in their relative levels might represent putativebiomarkers of disease progression (26).From these long lists of proteins identified in normal and

cancer cells, can we find those that may contribute to cancerdevelopment? The answer is likely yes, when considering thenature of many of the proteins identified that are predictedto be involved in cell cycle regulation, signal transduction,and proteolysis. For example, checkpoint kinase 1, a serine/threonine protein kinase that is a key mediator in the DNAdamage– induced checkpoint network (27), was highlydetected in normal epithelia but poorly detected in HNSCCtumors. Similarly, Apc, the protein product of the adenomatouspolyposis coli (apc) gene that prevents aberrant activity of theWnt/h-catenin signaling system and is the most frequentlymutated molecule in colon cancer (28), was detected as a singlepeptide only in normal oral epithelium. Low-density lipopro-tein receptor–related protein 12, also known as suppressor oftumorigenicity protein 7, a tumor suppressive protein whosegene is located on human chromosome 8 q22.2-23.1, a locus ofhigh polymorphism and genetic alterations in cancer biopsiesincluding HNSCC (29), was only found in normal oralepithelium. Similarly, a single peptide for cyclin K, a proteinthat acts as a regulatory subunit of CDK9 thereby regulating thetranscription of a subset of genes (30), was detected only innormal tissues. As cyclin K is regulated by p53, its loss in tumortissues may reflect the decreased p53 activity that characterizesHNSCC (3). Aligned with this possibility, individual peptidesfor a tumor suppressor gene on 17p13.3, hypermethylatedin cancer 1, HIC-1 , and a direct target for p53 that is involvedin the inhibition of cell growth and the initiation of cell deathand senescence programs in response to DNA damage (31),were identified in normal epithelial cells but only in one case ofPD tumor sample. Thus, a DNA damage sensing molecule,checkpoint kinase 1, at least two p53 targets, cyclin K andHIC-1, as well as lipoprotein receptor–related protein 12 andApc, the latter a well-known tumor suppressor protein poorlyinvestigated in HNSCC, seem to be more prominent in normalepithelial than in tumor cells. Collectively, these results suggestthe existence of a network of tumor-restricting mechanisms thatprotect the integrity of the normal squamous epithelium whoseloss or decreased expression and function may contribute toHNSCC progression.On the other hand, several proteins involved in cell cycle

progression, particularly G2-M transition and mitosis, such asseptin 9 and centromeric protein E, were only detected intumor samples, reflecting their active state of proliferation. Anunusual cell cycle regulating protein, prohibitin, which hasbeen recently observed to play an unexpected function in theactivation of Raf/MEK/ERK pathway by Ras and in modulatingepithelial cell adhesion and migration (32), was only detectedin tumors. Another surprising finding was the detection of twopeptides derived from EVI-5 oncogene. This protein was firstidentified in experimental T-cell lymphomas by retroviralinsertion strategies and has been recently shown to act togetherwith Polo-like kinase to ensure mitotic fidelity (33). Thus, bothprohibitin and EVI-5 may represent excellent candidates to playa role in aberrant cell growth in HNSCC.

Several molecules with a role in the transduction ofproliferative signals were also identified in normal and tumorHNSCC cells. For example, epidermal growth factor receptorwas detected in tumor samples but not in normal oralepithelial tissues, reflecting the overexpression of this growthfactor tyrosine kinase receptor in HNSCC (34). We also foundexpression of one epidermal growth factor receptor ligand,neuregulin-2, in HNSCC, suggesting an increased complexityof the epidermal growth factor receptor network in squamouscarcinogenesis. Similarly, numerous signaling molecules in-volved in cell migration were detected in HNSCC cells. Theyinclude several members of the Rho family of small GTPases,and few peptides derived from two novel guanine nucleotide

Fig. 2. Validation of biomarkers by immunohistochemistry on archival HNSCCtissues. A, archival tissues consisting normal and tumor HNSCC were processedand used for immunodetection of the indicated proteins with appropriateantibodies as described in Materials and Methods.

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exchange factors for Rho GTPases, RhoGEF 10 and RhoGEF19, in tumor cells, all suggestive of an important role for RhoGTPases and their regulatory proteins and downstream targetsin HNSCC progression. Two cell surface receptors, integrin h4

and plexin B1, which are involved in cell motility were alsoreadily detected in tumor cells. In this regard, whereas integrinh4 contributes to keratinocyte cell migration and facilitatestumor angiogenesis (35), plexin B1, which was initiallyidentified based on its role in axons guidance, is now knownto play an important role in endothelial cell migration andtumor angiogenesis (36). We have also shown that HNSCCexpress high levels of the plexin B1 ligand, semaphorin 4D(37), suggesting the existence of an autocrine plexin B1–semaphorin 4D loop that may promote HNSCC cell migra-tion. These proteins may also promote aberrant HNSCCgrowth, as suggested by recent studies indicating that integrinh4 and plexin B1 can stimulate members of the epidermalgrowth factor receptor and Met family of growth factorreceptors (38–40).

Proteases and their inhibitors form a complex proteolyticsystem and are ultimately responsible for cancer cell invasionand metastasis. In this study, the abundance of members of theADAMTS proteases family was particularly notable. Theseproteases likely contribute to extracellular matrix degradation,cell-to-cell adhesion, cell proliferation, and migration, and theprocessing of cytokines and growth factors, all aiding tumorprogression and angiogenesis. Cathepsin D, which is alysosomal aspartic protease, was also detected in the samples.Although cathepsins are involved in bulk protein turnover, theyalso have specialized roles in processes such as growth factorturnover and antigen presentation (41, 42). Cathepsin D wasthe most detectable protease in HNSCC samples, particularly inMD tumor cells. This cathepsin is often observed to be largelyoverexpressed in breast cancer tissues and their derived celllines and its expression levels correlate with the incidence ofclinical metastasis and shorter survival times (43). Furthermore,cathepsin D overexpression increases the growth and metastaticpotential of different cancer cells in vivo (43). Thus, emerging

Fig. 2 Continued. B, analysis of HNSCC biomarkers in HNSCC-specificTMA. Head and neck ^ specificTMAs consisting of control andWD, MD, and PDHNSCC tumorsamples were stained for indicated proteins with appropriate antibodies as described in Materials and Methods. RepresentativeTMA cores are depicted (top). StainedTMAswere ‘‘scored’’ based on tissue differentiation and staining intensity. For cytokeratin 4 and cytokeratin16, light gray represents >5% and <25% of cells stained; mid gray,26% to 50% of the cells stained; darkgray, 51% to 75%; andblack,76% to100% of the cells stained. For vimentin and desmoplakin, the percentage of positive tumors for eachstage of differentiation (black box) compared with negative (white box) is depicted. In each case, the number of nonneoplastic (Normal) tissues analyzed was10, and thenumber of HNSCC cancer tissues is indicated.

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evidence and our present findings suggest that this proteasemay play an unsuspected role in HNSCC progression.Taken together, the ability to combine laser capture

microdissection and in-depth proteomic analysis of formalin-fixed, paraffin-embedded tissues provides a wealth of informa-tion regarding the nature of the proteins expressed in normalsquamous epithelium and tumor progression. These proteinsinclude a number of tumor suppressor molecules and micro-RNA processing proteins likely involved in protecting theintegrity of the cellular genome in normal epithelial cells andtheir resident progenitor stem cells, as well as many moleculesinvolved in aberrant cell proliferation, survival, angiogenesis,proteolysis, and migration, whose contribution to tumorgrowth, resistance to treatment, and the metastatic spread ofHNSCC can now begin to be explored. The emerginginformation has also enabled identification of a large numberof proteins that are differentially expressed in normal oralsquamous epithelia and tumors exhibiting distinct differentia-

tion characteristics, thus representing suitable makers to studytumor progression. The future evaluation of the tumor makersdescribed in this study may afford an opportunity to exploretheir diagnostic and prognostic value, in particular for the earlydetection of HNSCC. On the other hand, we expect that ourstudy, documenting the successful use of proteomic techniquesand bioinformatic tools to analyze molecules expressed inarchival tumor and normal tissues, may now provide a proof ofprinciple that will boost ongoing systems to increase the scaleof data-generating proteomic efforts, which may ultimately leadto discovery of novel clinically relevant biomarkers andtherapeutic targets for HNSCC and other human malignancies.

Acknowledgments

We thank Thomas H. Bugge, Ana Raimondi, Panomwat Amornphimoltham,Chidchanok Leethanakul, RobertT. Dorsam,Thomas Guiel, and Marlene Darfler fortheir technical help, advice, and guidance.

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2008;14:1002-1014. Clin Cancer Res   Vyomesh Patel, Brian L. Hood, Alfredo A. Molinolo, et al.   ProgressionTissues: A Molecular Portrait of Head and Neck Cancer Proteomic Analysis of Laser-Captured Paraffin-Embedded

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